CHAPTER 1

Overview of the Zebrafish System H. William Detrich, III,* Monte WesterfieldYt and Leonard I. ZonS * Department of Biology Northeastern University Boston, Massachusetts 021 15

t Institute of Neuroscience University of Oregon Eugene, Oregon 97403

Howard Hughes Mechcal Institute Children’s Hospital Boston. Massachusetts 021 15

I. Introduction 11. History of the Zebrafish System and Its Advantages and Disadvantages

111. Cell and Developmental Biology, Organogenesis, and Human Disease IV. Genetics and Genomics V. Future Prospects

VI. Conclusion References

I. Introduction

A central dogma of developmental biology today is that the fundamental genetic mechanisms that control the development of metazoans have been con- served evolutionarily, albeit frequently modified in their application. For exam- ple, invertebrates and vertebrates employ homologous signaling systems that act antagonistically to establish topologically equivalent but spatially reversed, dorsal-ventral axes (De Robertis and Sasai, 1996). Based on mutant phenotype and protein structure, vertebrate ventralizing signals (e.g., bone morphogenetic proteins BMP-2 and BMP-4) are functionally homologous to Drosophilu Deca- pentaplegic, which functions in dorsal determination in the fly, and the vertebrate

METHODS IN CELL BIOLOGY, VOL. 59 Copyright 0 1999 by Academic Press. All lights of reproduction in any form reserved. 0091-679X/99 830.00

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dorsalizer Chordin is homologous to the Drosophilu ventralizing signal, Short gastrulation. Nevertheless, some aspects of development are uniquely vertebrate. The neural crest, for example, is a group of migratory cells that arises in the embryo at the border between neural and nonneural ectoderm. These cells move to many regions of the embryo to form numerous tissues, including part of the cranial skeleton and the peripheral nervous system. The development of complex organ systems, such as the brain, heart, and kidneys, is another hallmark of vertebrates that is not easily studied in invertebrate genetic systems. For develop- mental analysis of vertebrates, the zebrafish, Dunio rerio, has arguably emerged as the genetic system par excellence.

In December 1996, the world of biological science witnessed the equivalent of Yogi Berra’s ‘‘ddjja vu all over again.” That month’s issue of the journal Development was devoted entirely to the description, in 37 articles, of approxi- mately 2000 mutations that perturb development of the zebrafish (for highlights, see Currie, 1996; Eisen, 1996; Grunwald, 1996; Holder and McMahon, 1996). This magnificent accomplishment, the result of two independent large-scale mutagenic screens of the zebrafish genome and phenotypic analysis of embryonic develop- ment in the mutants obtained, approximates in a vertebrate the earlier saturation mutagenic screen in Drosophilu (Niisslein-Volhard and Wieschaus, 1980). In- deed, two of the investigators leading the zebrafish screens, Christiane Niisslein- Volhard of the Max-Planck-Institut fur Entwicklungsbiologie in Tiibingen and Wolfgang Driever of the Massachusetts General Hospital (MGH) in Boston, were veterans of the Drosophilu program. Working at the European Molecular Biology Laboratory in Heidelberg, “Janni” Nusslein-Volhard and her colleague Eric Wieschaus (co-recipients with Edward Lewis of the 1995 Nobel Prize in Physiology or Medicine) conducted the now legendary Drosophilu screen, and Driever, as a later member of the Nusslein-Volhard laboratory, analyzed many of the mutants to determine the essential signaling pathways that control develop- ment of the fly’s body plan. Niisslein-Volhard in Tubingen, and Driever and his colleague Mark Fishman at the MGH, subsequently applied the conceptual framework of the Drosophilu screen to the fish. The community of developmental biologists owes these three individuals and their many colleagues and collabora- tors a tremendous debt of gratitude for this repeat performance.

11. History of the Zebrafish System and Its Advantages and Disadvantages

These recent mutagenesis screens provided proof-of-principle that classical, forward genetics can be used to understand vertebrate development. The identi- fication and study of mutations has been extraordinarily successful in providing an understanding of the early development of Drosophilu and of the nematode worm, Cuenorhubditis eleguns. However, the same level of analysis of early developmental events in vertebrates has been more problematic. In the mouse,

1. Overview of the Zebrafish System 5

historically the species of choice for studies of vertebrate developmental genetics, much of embryogenesis is difficult to follow because it occurs within the mother’s uterus. Beginning about 20 years ago at the University of Oregon, George Strei- singer recognized the power of genetic analysis for understanding development and the advantages of a small tropical fish with external fertilization as a verte- brate for this approach. Streisinger selected the zebrafish, a freshwater fish com- monly available in pet stores, because it has a relatively short generation time (2-3 months), produces large clutches of embryos (100-200 per mating), and provides easy access to all developmental stages. Zebrafish embryos are optically transparent throughout early development, which facilitates a host of embryologi- cal experiments and the rapid morphological screening of the live progeny of mutagenized fish for interesting mutations. Before his untimely death in 1984, Streisinger’s group cloned the zebrafish (Streisinger et al., 1981) and developed techniques for mutagenesis (e.g., Walker and Streisinger, 1983; Grunwald and Streisinger, 1992a; 1992b), genetic mapping (Streisinger et al., 1986), and clonal analysis of development by genetic mosaics (Streisinger et al., 1989). They also used F1 screens of mutagenized fish to isolate zygotic recessive lethal mutations with wonderfully curious embryonic phenotypes (Grunwald et aL, 1988; Felsen- feld et aL, 1991).

Streisinger’s discoveries, as well as his enthusiasm and generosity, stimulated a number of other laboratories to begin using the zebrafish for developmental and genetic studies. Initially, all of these laboratories were also in Oregon. These groups have extended Streisinger’s original studies by isolating and analyzing additional informative mutants (Kimmel, 1989; Kimmel et al., 1989; Hatta et al., 1991; Halpern et al., 1993,1995) and have developed techniques for the produc- tion of transgenic zebrafish (Stuart et al., 1988; Westerfield et al., 1993). Moreover, recent work has demonstrated the advantages of zebrafish for cellular studies of vertebrate embryonic development. The embryo is organized very simply (Kim- me1 et al., 1995) and has fewer cells than other vertebrate species under investiga- tion (Kimmel and Westerfield, 1990). Its transparent cells are accessible for manipulative study. For example, cells can be injected with tracer dyes in intact developing embryos to track emerging cell lineages (Kimmel and Warga, 1986) or axons growing to their targets (Eisen et al., 1986). Uniquely identified young cells can be ablated singly (Eisen et aL, 1989) or transplanted individually to new positions (Eisen, 1991) to address the positional influences on development at a level of precision that is unprecedented in any species. The combination of easy mutagenesis and powerful phenotypic screens of the earliest developmental stages eliminates, in principle, the biased detection of mutant phenotypes ob- served in the mouse, where the scoring of mutants is generally restricted to neonatal and adult animals due to the intrauterine development of the embryos. The more recent advent of tools for mapping mutations and candidate genes in the zebrafish genome has already begun to facilitate the isolation and functional analysis of genes required for normal development. Even small laboratories can

6 H. William Detrich, 111, et al.

conduct reasonably sized screens for new mutations, and the cost of a fish facility necessary to support such research is significantly lower than for the mouse.

Several disadvantages of the zebrafish system are also apparent. We presently lack in the zebrafish system methods to generate embryonic stem cells for gene “knock-outs” by homologous recombination. In the absence of such methods, we envision a cooperative and synergistic game of “ping pong” between the zebrafish and mammalian research communities. Knock-out analysis of the mouse homologues of genes identified via the study of zebrafish mutations should lead to a greater understanding of gene function in vertebrate development. Conversely, conserved syntenies between mammalian and zebrafish genomes, and the numer- ous mammalian-expressed sequence tags (ESTs), will continue to provide candi- date genes for zebrafish mutations.

Another potential disadvantage is genetic redundancy in the zebrafish genome, which probably results from duplication of the fish genome subsequent to the phylogenetic divergence of fish and mammals (Chapter 8, Vol. 60). This redun- dancy may complicate the comparison of homologous developmental pathways in these taxa. Alternatively, extra gene copies may simplify some types of analysis because complex functions in mammals may have been separated and allocated to different gene paralogues in fish.

111. Cell and Developmental Biology, Organogenesis, and Human Disease

The zebrafish embryo (Chapter 2) provides numerous opportunities to examine cellular processes in early development. For example, one can culture cells from embryos (Chapters 3-4) in vitro for mechanistic studies of cell signaling pathways, analyze gene and protein expression in situ (Chapter 6), perturb development using physical and chemical treatments or by ectopic expression of dominant- negative proteins (Herskowitz, 1987; Chapters 7-8), and assay lineage commit- ment by explant assay (Chapter 9). The roles of cell movements and the cytoskele- ton in embryonic axis formation are particularly amenable to analysis (Chapters 10-13). Given that some of these processes occur prior to the activation of the zygotic genome, maternal-effect mutants may prove to be especially informative in revealing the molecular players.

The analysis of vertebrate organogenesis has always been problematic. Thanks to the recent zebrafish genetic screens, mutations that affect virtually all major organ systems are now available for phenotypic and molecular characterization (Chapters 14-20). The hematopoietic mutants, for example, comprise 26 distinct complementation groups that perturb development of the erythroid lineage from the earliest stages of stem cell commitment to terminal differentiation (Ransom et al., 1996; Weinstein et al., 1996; Chapter 17). The cardiovascular mutants include some that affect early development of the heart, vasculature, and blood and others that disrupt the function of an otherwise morphologically normal

1. Overview of the Zebrafish System 7

organ system (Chapters 17, 19). Mutations that interfere with development of the central nervous system (Chapters 14, 20), the retina (Chapter 15), and fins (Chapter 16) are also plentiful. We can anticipate a rich harvest of information from study of these mutants.

Zebrafish mutants will also provide useful models of human diseases. The one- eyed pinhead mutation, which disrupts an EGF-signaling pathway in zebrafish (Zhang et al., 1998), phenocopies the human condition holoprosencephaly. grid- lock, which fails to develop trunk vasculature, resembles the human condition coarctation of the aorta, a common and lethal birth defect (Weinstein et al., 1995). The hematopoietic mutants include representatives of thalassemias, por- phyrias, and other human conditions (Chapter 17).

IV. Genetics and Genomics

Although highly successful, the large-scale zebrafish mutant screens (cf. Chap- ter 2, Vol. 60) failed to achieve saturation. Currie (1996) estimates that the degree of saturation obtained by the combined Tubingen and MGH screens ranges from 50-90% of the genes detectable by the methods employed. Further- more, the morphological parameters of the screens probably precluded the identi- fication of many interesting mutants. Thus, it is likely that many investigators will now perform additional screens targeted to particular developmental pro- cesses. One exquisite example is the retinotectal projection screen carried out by Friedrich Bonhoeffer and his laboratory (Baier et al., 1996; Karlstrom et al., 1996; Trowe et al., 1996) in conjunction with the Tubingen screen of the Nusslein- Volhard laboratory. A second is the screen for neural crest mutations (Henion et al., 1996). Maternal effects screens (Chapter 1, Vol. 60) are in progress. The Oregon laboratories have initiated screens based on RNA in situ hybridization. Nancy Hopkins (MIT) and her laboratory are now conducting a large-scale insertional mutagenesis screen (Chapter 5, Vol. 60), which promises to ease genetic analysis by providing convenient molecular tags for isolating disrupted genes (Gaiano et al., 1996). Finally, transposable elements (Chapter 6, Vol. 60) and transgenesis with cell-specific promoters that drive the expression of green fluorescent protein (Chapter 7, Vol. 60) (Jessen et al., 1998) will provide impor- tant tools for genetic analysis.

Genomic methodologies are advancing rapidly in the zebrafish system. We now have high-density genetic maps (Chapter 8, Vol. 60) that incorporate a variety of markers: randomly amplified polymorphic DNAs (RAPDs) (Chapter 9, Vol. 60), simple-sequence length polymorphisms (e.g., CA repeats) (Chapter 10, Vol. 60; Knapik et al., 1998); single-strand conformational polymorphisms (SSCPs) (Chapter 11, Vol. 60) amplified fragment length polymorphisms (AFLPs) (Chapter 12, Vol. 60), and expressed sequence tags (ESTs) (Chapter 13, Vol. 60). Large-insert yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), and P1 artificial chromosome (PAC) libraries have been

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produced (Chapter 14, Vol. 60) and are commercially available. With these tools and techniques, we can now map mutations and apply candidate or positional cloning (Chapter 15, Vol. 60) strategies to recover the disrupted genes. At the Third Cold Spring Harbor Conference on Zebrafish Development and Genetics, researchers reported the genes for approximately 20 mutants, two identified via positional cloning and the remainder by the candidate approach. Other genomic tools in development include radiation hybrid panels (Chapter 16, Vol. 60), somatic cell hybrids (Chapter 17, Vol. 60); fluorescent in situ hybridization, (FISH) (Chapter 18, Vol. 60); and a multipurpose zebrafish database (Chapter 19, Vol. 60).

V. Future Prospects

What does the future hold for the zebrafish system? Molecular, cellular, and developmental studies of the extant mutant collections should yield a wealth of new knowledge regarding vertebrate embryogenesis. We envision many more mutant screens directed at particular developmental processes and employing molecular probes (e.g., antibodies, antisense RNAs) for phenotypic analysis. Furthermore, the genetics of behavior will certainly be tackled. The genetic epistasis analysis of double mutants will help to establish molecular signaling pathways. The application of suppressor and enhancer screening strategies should reveal gene interactions, and the generation of conditional mutations will contrib- ute to a temporal dissection of gene function. We predict that the zebrafish, with its present and future methodologies and infrastructure, will make important and probably surprising contributions to our understanding of the vertebrate development program.

VI. Conclusion

The consensus of the biologists in attendance at the Third Cold Spring Harbor Conference on Zebrafish Development and Genetics is that the zebrafish has now “arrived” as a viable compelling genetic system for study of vertebrate development. Novel contributions by the zebrafish system have been made and will continue to be made at an accelerating rate. These two volumes provide a solid compilation of current methods in zebrafish development and genetics. We trust that they will stimulate further technical development and will attract new scientific converts to the system. Now is certainly the time to fish-not to cut bait.

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